Novel Nile Blue derivatives as fluorescent probes for DNA

Article abstract of DOI:10.1016/j.dyepig.2013.05.007

Cationic dyes based on benzo[a]phenoxazinium chlorides either mono- or disubstituted with ethyl, octyl and dodecyl groups at the amino side chain at the 9-position of the tetracyclic ring, and possessing a propyl group at the 5-amino position were synthesised, characterised and used in photophysical studies with DNA and in agarose gel electrophoresis assays. Photophysical results showed that compounds with diethyl and octyl substituents intercalated in DNA above a phosphate dye ratio of 10. All compounds associated with DNA as evidenced by their use as coloured markers of migrating DNA bands in agarose gel electrophoresis experiments.

Full text of DOI:10.1016/j.dyepig.2013.05.007

Accepted Manuscript
Novel Nile Blue derivatives as fluorescent probes for DNA
B. Rama Raju, Sarala Naik, Paulo J.G. Coutinho, M.Sameiro T. Gonç alves
PII:
S0143-7208(13)00172-1
10.1016/j.dyepig.2013.05.007
DYPI 3944
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 13 March 2013
Revised Date: 6 May 2013
Accepted Date: 7 May 2013
Please cite this article as: Rama Raju B, Naik S, Coutinho PJG, Gonç alves MST, Novel Nile
Blue derivatives as fluorescent probes for DNA, Dyes and Pigments (2013), doi: 10.1016/
j.dyepig.2013.05.007.
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ACCEPTED MANUSCRIPT
Novel Nile Blue derivatives as fluorescent probes for DNA
B. Rama Raju, Sarala Naik, Paulo J. G. Coutinho and M. Sameiro T. Gonçalves*
Highlights
•
•
•
New NIR cationic dyes based on benzo[a]phenoxazinium chlorides were
synthesised.
The dyes showed intercalative behaviour, aggregation or groove binding in
DNA.
They display promising capabilities as DNA colored markers in electrophoresis
assays.

ACCEPTED MANUSCRIPT
GRAPHICAL ABSTRACT
Novel Nile Blue derivatives as fluorescent probes for DNA
B. Rama Rajua,b, Sarala Naika,b, Paulo J. G. Coutinhob and M. Sameiro T. Gonçalvesa*
aCentre of Chemistry, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal
bCentre of Physics, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal
A
new
set
of
NIR
chlorides
fluorescent
was
benzo[a]phenoxazinium
synthesised. Photophysical studies and agarose
gel electrophoresis experiments with these
Nile Blue derivatives revealed their potential
as DNA markers and probes.
1

ACCEPTED MANUSCRIPT
Novel Nile Blue derivatives as fluorescent probes for DNA
B. Rama Rajua,b, Sarala Naika,b, Paulo J. G. Coutinhob and M. Sameiro T. Gonçalvesa*
aCentre of Chemistry, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal
bCentre of Physics, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal
Abstract: Cationic dyes based on benzo[a]phenoxazinium chlorides either mono- or
disubstituted with ethyl, octyl and dodecyl groups at the amino side chain at the 9-position of
the tetracyclic ring, and possessing a propyl group at the 5-amino position were synthesised,
characterised and used in photophysical studies with DNA and in agarose gel electrophoresis
assays. Photophysical results showed that compounds with diethyl and octyl substituents
intercalated DNA above a phosphate dye ratio of 10. All compounds associated with DNA as
evidenced by their use as colored markers of migrating DNA bands in agarose gel
electrophoresis experiments.
Keywords: Nile Blue; Benzo[a]phenoxazinium dyes; DNA probes; Near-infrared probes;
Cationic dyes
1. Introduction
In the recent years, labeling of biological matter such as amino acids, proteins and cells
with synthetic fluorescent reagents has become an indispensible tool in bioanalytical sciences
[1,2]. Among the fluorescent reagents, phenoxazine core based molecules are extensively
studied due to their profound applications in terms of extra stability and brightness [3-5]. Nile
Blue (NB), a phenoxazine dye, contains a planar rigid aromatic system which enables the
intercalation into the non-planar interior of the DNA helix [6]. Due to this intercalation binding
[7], NB and derivatives were used as fluorescent labels in therapeutic [8-10] and scientific areas
[11-14].
NB in comparison with other conventional intercalators proved to be superior mainly
due to low toxicity and good sensitivity for DNA quantification purposes [6]. In addition, NB
was used as marker for the detection of DNA in electrophoresis gels [15]. The intercalation of
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small organic molecules or ligands with nucleic acids is mainly associated with π–stacking
[16]. Despite the importance and application of NB as molecular fluorescent probe, limited
research has been done with new derivatives of the benzo[a]phenoxazinium dye family. In
continuation of our earlier observations, combined with synthetic conciseness [17-20] and
evaluation of the potential of this family of fluorophores as DNA labels [21,22], we
synthesized new benzo[a]phenoxazinium chlorides either mono- or disubstituted with different
alkyl groups at the amino side chain at the 9-position of the tetracyclic ring, and having the
propyl group at the 5-amino position. The photophysical behavior of these new Nile Blue
derivatives in the presence of DNA was evaluated and also electrophoresis experiments in
agarose gel were performed in order to assess their potential as DNA markers.
2. Experimental section
2.1. Material and instruments
All melting points were measured on a Stuart SMP3 melting point apparatus. TLC analysis was
carried out on 0.25 mm thick precoated silica plates (Merck Fertigplatten Kieselgel 60F254) and
spots were visualised under UV light. Chromatography on silica gel was carried out on Merck
Kieselgel (230-240 mesh). IR spectra were determined on a BOMEM MB 104
spectrophotometer. NMR spectra were obtained on a Varian Unity Plus Spectrometer at an
operating frequency of 300 MHz for 1H and 75.4 MHz for 13C or a Bruker Avance III 400 at an
1
operating frequency of 400 MHz for H and 100.6 MHz for 13C using the solvent peak as
internal reference at 25 ºC. All chemical shifts are given in ppm using δH Me4Si = 0 ppm as
reference, and J values are given in Hz. Assignments were made by comparison of chemical
shifts, peak multiplicities and J values and were supported by spin decoupling-double
resonance and bidimensional heteronuclear correlation techniques. Low and high resolution
mass spectrometry analyses were performed at the “C.A.C.T.I. - Unidad de Espectrometria de
Masas”, at University of Vigo, Spain. Absorption spectra (200-800 nm) were obtained using
Shimadzu UV/3101PC spectrophotometer and fluorescence spectra with a Spex Fluorolog
spectrofluorometer. Commercially available reagents and solvents were used without further
purification.
2.2. Synthetic methods for the preparation of precursors 2-4
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2.2.1. 5-(Dioctylamino)-2-nitrosophenol hydrochloride 2.
To an ice-cold solution of 3-(dioctylamino)phenol (0.333 g, 1 mmol) in ethanol (1 mL),
concentrated hydrochloric acid (0.21 mL) was added and stirred during 5 min. The solution of
sodium nitrite (0.076 g, 1.1 mmol) in water (0.1 mL) was then added drop-wise within an
interval of 20-25 min. The resulting mixture was stirred for 3 h and monitored by TLC
(dichloromethane/methanol, 9.5:0.5 and 9:1 vol/vol). After evaporation of the reaction,
compound 1 was obtained as an orange oily solid (0.399 g) and was used in the following step
without any purification.
3-(Octylamino)phenol and 3-(dioctylamino)phenol
To a solution of 3-aminophenol (1.0 g, 9.10 mmol) in ethanol (6 mL), 1-bromooctane (1.86
mL, 10.8 mmol) was added and the reaction mixture was refluxed for 18.5 h. The progress of
the reaction was monitored by TLC which showed the presence of two products. After
purification by column chromatography on silica gel with dichloromethane and
dichloromethane/methanol (mixtures of increasing polarity), as the eluent, 3-
(octylamino)phenol was obtained as a brown oil (1.018 g, 51%). TLC (dichloromethane): Rf =
1
0.21. H NMR (400 MHz, CDCl3): δ = 0.84 (t, J = 7.2 Hz, 3 H, NH(CH2)7CH3), 1.10-1.22 (s,
10 H, NHCH2CH2(CH2)5CH3), 1.72-1.85 (m, 2 H, NHCH2CH2(CH2)5CH3), 3.26 (t, J = 8.4 Hz,
2 H, NHCH2CH2(CH2)5CH3), 6.84 (dd, J = 8.2 and 2.0 Hz, 1 H, 4-H), 6.99 (dd, J = 8.2 and
1.2 Hz, 1 H, 6-H ), 7.14 (t, J = 8.4 Hz, 1 H, 5-H), 7.20 (t, J = 2.0 Hz, 1 H, 2-H), 7.83 (br s, 2 H,
NH, OH). 13C NMR (100.6 MHz, CDCl3): δ = 14.00 (NH(CH2)7CH3), 22.50 (CH2), 25.46
(NHCH2CH2(CH2)5CH3), 26.50 (CH2), 28.88 (CH2), 29.00 (CH2), 31.60 (CH2), 52.60
(NHCH2CH2(CH2)5CH3), 109.77 (C-2), 113.79 (C-6), 116.99 (C-4), 130.98 (C-5), 135.54 (C-
3), 157.61 (C-1). IR (Neat): νmax = 3261, 2956, 2927, 2857, 2778, 2467, 1621, 1601, 1576,
1503, 1485, 1468, 1427, 1378, 1316, 1289, 1223, 1152, 1082, 1000, 959, 861, 782, 737, 688,
666 cm-1. HRMS: m/z (TOF EI): calcd. for C14H23NO [M+]: 221.1780; found: 221.1784.
In addition to the 3-(octylamino)phenol, 3-(dioctylamino)phenol was also obtained as a brown
1
oil (0.712 g, 24%). TLC (dichloromethane): Rf = 0.56. H NMR (CDCl3, 300 MHz): δ = 0.90
(t, J = 6.9 Hz, 6 H, (N((CH2)7CH3)2), 1.28 (br s, 20 H, N(CH2CH2(CH2)5CH3)2), 1.58 (br s, 4
H, N(CH2CH2(CH2)5CH3)2), 3.24 (t, J = 7.8 Hz, 4 H, N(CH2CH2(CH2)5CH3)2), 6.38 (br s, 3 H,
2-H, 4-H, 6-H), 7.09 (t, J = 7.8 Hz, 1 H, 5-H). 13C NMR (75.4 MHz, CDCl3): δ = 14.04
(2×CH3), 22.58 (2×CH2), 26.75 N(CH2CH2(CH2)5CH3)2), 27.00 (2×CH2), 29.24 (2×CH2),
29.35 (2×CH2), 31.75 (2×CH2), 52.30 (N(CH2CH2(CH2)5CH3)2), 100.82 (C-2), 105.93 (C-6, C-
4), 130.15 (C-5), 148.18 (C-3), 157.01 (C-1). IR (Neat): νmax = 3299, 2955, 2926, 2855, 2726,
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1619, 1579, 1504, 1467, 1400, 1370, 1276, 1241, 1221, 1167, 1142, 1114, 1013, 999, 832, 821,
753, 723, 688 cm–1. HRMS: m/z (TOF EI): calcd. for C22H39NO [M+]: 333.3032; found
333.3034.
2.2.2. 2-Nitroso-5-(octylamino)phenol hydrochloride 3
Starting from 3-(octylamino)phenol (0.336 g, 1.52 mmol) in ethanol (2 mL) and concentrated
hydrochloric acid (0.40 mL), using sodium nitrite (0.115 g, 1.67 mmol) in water (0.1 mL), and
following the same procedure as described before for the preparation of 1, compound 2 was
obtained as an orange oily solid (0.436 g) and was used in the following step without any
purification.
2.2.3. 5-(Dodecylamino)-2-nitrosophenol hydrochloride 4
Starting from 3-(dodecylamino)phenol (0.416 g, 1.5 mmol) in ethanol (2 mL) and concentrated
hydrochloric acid (0.40 mL), using sodium nitrite (0.114 g, 1.65 mmol) in water (0.1 mL), and
following the same procedure as described before for the preparation of 1, compound 2 was
obtained as an orange oily solid (0.515 g) and was used in the following step without any
purification.
3-(Dodecylamino)phenol
To a solution of 3-aminophenol (2.0 g, 0.183 mmol) in ethanol (6 mL), 1-bromododecane (6.5
mL, 0.273 mmol) was added and the reaction mixture was refluxed for 10 h. After purification
by column chromatography on silica gel with dichloromethane and dichloromethane/methanol,
mixtures of increasing polarity, as the eluent, 3-(dodecylamino)phenol was obtained as a brown
solid (2.931g, 58%). TLC (dichloromethane): Rf = 0.17. 1H NMR (CDCl3, 400 MHz):
δ 0.87 (t,
J = 7.2 Hz, 3 H, NH(CH2)11CH3), 1.10-1.35 (m, 18 H, NHCH2CH2(CH2)9CH3), 1.70-1.90 (m, 2
H NHCH2CH2(CH2)9CH3), 3.26 (t, J = 8.4 Hz, 2 H, NHCH2CH2(CH2)9CH3), 6.82 (dd, J = 8.4
and 1.6 Hz, 1 H, 4-H), 6.98 (dd, J = 8.0 and 1.2 Hz, 1 H, 6-H), 7.14 (t, J = 8.0 Hz, 1H, 5-H),
13
7.22 (br s, 1 H, 2-H), 8.4 (br s, 2 H, NH, OH). C NMR (CDCl3, 100.6 MHz):
δ = 14.07
(NH(CH2)11CH3), 22.64 (CH2), 25.55 (NHCH2CH2(CH2)9CH3), 26.57 (CH2), 29.00 (CH2),
29.31 (CH2), 29.43 (CH2), 29.49 (CH2), 29.58 (CH2), 29.59 (CH2), 31.87 (CH2), 52.54
(NHCH2CH2(CH2)9CH3), 109.69 (C-2), 113.72 (C-6), 116.92 (C-4), 130.97 (C-5), 135.67 (C-
3), 157.65 (C-1). FTIR (KBr 1%): νmax 3282, 2918, 2851, 2730, 2630, 2600, 2414, 1619, 1571,
1499, 1484, 1468, 1417, 1382, 1338, 1318, 1288, 1250, 1237, 1216, 1176, 1153, 1133, 1087
cm-1. HRMS: m/z (EI): calcd. for C18H31NO [M+]: 277.2406; found: 277.2410.
5

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In addition to the 3-(dodecylamino)phenol, 3-(didodecylamino)phenol was also obtained as a
1
purple solid (1.598g, 20%). TLC (dichloromethane): Rf = 0.53. H NMR (CDCl3, 400 MHz):
δ
= 0.85 (t, J = 7.2 Hz, 6 H, N((CH2)11CH3)2), 1.10-1.40 (m, 36 H, N(CH2CH2(CH2)9CH3)2),
1.92 (br s, 4 H, N(CH2CH2(CH2)9CH3)2), 3.29 (br s, 2 H, NCH2CH2(CH2)9CH3), 3.47 (br s, 2
H, NCH2CH2(CH2)9CH3), 7.16 (br s, 2 H, 4-H, 6-H), 7.28 (t, J = 8.0 Hz, 1 H, 5-H), 7.45 (br s,
1 H, 2-H), 8.68 (br s, 1 H, OH). 13C NMR (CDCl3, 100.6 MHz):
δ = 14.03 ((N(CH2)11CH3)2),
22.59 (2×CH2), 24.77 (N(CH2CH2(CH2)9CH3)2), 26.46 (2×CH2), 28.93 (2×CH2), 29.24
(2×CH2), 29.34 (2×CH2), 29.38 (2×CH2), 29.49 (2×CH2), 29.52 (2×CH2), 31.82 (2×CH2),
59.11 (N(CH2CH2(CH2)9CH3)2), 108.91 (C-2), 113.21 (C-6), 118.19 (C-4), 130.96 (C-5),
138.05 (C-3), 158.89 (C-1). FTIR (KBr 1%): νmax 3175, 3046, 2916, 2850, 2636, 2605, 2537,
1613, 1511, 1490, 1471, 1446, 1418, 1406, 1373, 1355, 1318, 1284, 1226, 1212, 1204, 1181,
1141, 1126, 1091, 1044 cm-1. HRMS: m/z (TOF EI): calcd. for C30H55NO [M+]: 445.4284;
found: 445.4283.
2.3. General procedure for the synthesis of benzo[a]phenoxazines 6-9
To a cold solution (ice bath) of 2-nitrosophenol hydrochloride (1.5 mmol) in methanol (3 mL),
N-propylnaphthalen-1-amine 5 (1 mmol) and concentrated hydrochloride acid (40 µL) were
added. The mixture was refluxed until the completion of the reaction and was monitored by
TLC with chloroform and mixtures of chloroform/methanol, as the eluent. The solvent was
evaporated and the crude mixture was purified by column chromatography on silica gel with
chloroform and chloroform/methanol, mixtures of increasing polarity, as the eluent. The
expected benzo[a]phenoxazines 6-9 were isolated as blue solids in good yields.
2.3.1.
N-Octyl-N-(5-(propylamino)-9H-benzo[a]phenoxazin-9-ylidene)octan-1-aminium
chloride 7
The product of the reaction of 5-(dioctylamino)-2-nitrosophenol hydrochloride 2 (0.399 g, 1.0
mmol) with N-propylnaphthalen-1-amine 5 (0.124 g, 0.67 mmol) (reflux time 14 h) was
chromatographed with chloroform and chloroform/methanol (mixtures of increasing polarity),
as the eluent, to give compound 7 (0.439 g, 78%). Mp = 150-152°C. TLC
(chloroform/methanol, 9.4:0.6): Rf = 0.20. 1H NMR (400 MHz, CDCl3): δ = 0.80-1.0 (m, 6 H,
NHCH2CH2CH3, N(CH2)7CH3), 1.11 (br s, 3 H, N(CH2)7CH3), 1.10-1.50 (br s, 20 H,
N(CH2CH2(CH2)5CH3)2), 1.72 (br s, 4 H, N(CH2CH2(CH2)5CH3)2), 1.98 (br s, 2 H,
NHCH2CH2CH3), 3.50 (br s, 4 H, N(CH2CH2(CH2)5CH3)2), 3.86 (br s, 2 H, NHCH2CH2CH3),
6.61 (br s, 2 H, 6-H, 8-H), 6.95 (br s, 1 H, 10-H), 7.70-7.90 (m, 3 H, 2-H, 11-H, 3-H), 8.86 (br
6

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s, 1 H, 4-H), 9.45 (br s, 1 H, 1-H), 11.90 (br s, 1 H, NH). 13C NMR (100.6 MHz, CDCl3): δ =
11.58 (N(CH2)7CH3), 14.00 (NHCH2CH2CH3, N(CH2)7CH3), 22.37 (2×CH2), 22.54
(NHCH2CH2CH3), 26.97 (2×CH2), 27.36 (N(CH2CH2(CH2)5CH3)2), 29.15 (2×CH2), 29.29
(CH2), 29.60 (CH2), 31.68 (2×CH2), 46.53 (NHCH2CH2CH3), 52.01 (N(CH2CH2(CH2)5CH3)2),
93.62 (C-6), 95.98 (C-8), 113.46 (C-10), 124.07 (C-4), 124.32 (Ar-C), 126.65 (C-1), 128.27
(Ar-C), 130.74 (Ar-C), 130.94 (C-3), 132.12 (C-2), 132.40 (C-11), 135.97 (Ar-C), 147.34 (Ar-
C), 151.63 (Ar-C), 153.00 (C-9), 158.92 (C-5). IR (KBr 1%) νmax = 3446, 3154, 3012, 2954,
2924, 2853, 1641, 1586, 1545, 1494, 1480, 1455, 1433, 1330, 1289, 1273, 1236, 1213, 1180,
1162, 1146, 1120, 1053, 1012, 1001, 972, 923, 915, 900, 886, 870, 855 cm-1. HRMS: m/z (EI):
calcd. for C35H50N3O [M++1]: 528.39426; found: 528.39484.
2.3.2. N-(5-(Propylamino)-9H-benzo[a]phenoxazin-9-ylidene)octan-1-aminium chloride 8
The product of the reaction of 2-nitroso-5-(octylamino)phenol hydrochloride 3 (0.436 g, 1.52
mmol) with N-propylnaphthalen-1-amine 5 (0.140 g; 0.75 mmol) (reflux time 8 h) was
chromatographed with chloroform and chloroform/methanol (mixtures of increasing polarity),
as the eluent, to give compound 8 (0.240 g, 35%). Mp = 165-168°C. TLC
1
(chloroform/methanol, 9:1): Rf = 0.45; H NMR (400 MHz, CDCl3): δ = 0.86 (t, J = 6.8 Hz, 3
H, NHCH2CH2CH3), 1.07 (t, J = 6.8 Hz, 3 H, NH(CH2)7CH3), 1.20-1.43 (m, 10 H,
NHCH2CH2(CH2)5CH3), 1.74 (br s, 2 H, NHCH2CH2(CH2)5CH3), 1.83-2.0 (m, 2 H,
NHCH2CH2CH3), 3.11 (br s, 2 H, NHCH2CH2(CH2)5CH3), 3.53 (br s, 2 H, NHCH2CH2CH3),
6.22 (s, 2 H, 6-H, 8-H), 7.15 (br s, 1 H, 10-H), 7.35 (d, J = 8.4 Hz, 1 H, 11-H), 7.68-7.86 (m, 2
H, 2-H, 3-H), 8.43 (br s, 1 H, NH), 8.59 (d, J = 7.6 Hz, 1 H, 4-H), 8.83 (br s, 1 H, 1-H), 10.35
(br s, 1 H, NH). 13C NMR (100.6 MHz, CDCl3): δ = 11.57 (NH(CH2)7CH3), 14.04
(NHCH2CH2CH3), 21.98 (NHCH2CH2CH3), 22.58 (CH2), 27.15 (CH2), 28.51 (CH2), 29.19
(NHCH2CH2(CH2)5CH3), 29.25 (CH2), 31.76 (CH2), 43.90 (NHCH2CH2(CH2)5CH3), 46.24
(NHCH2CH2CH3), 92.34 (C-6), 94.13 (C-8), 118.34 (C-10), 123.35 (C-4), 123.79 (C-1),
125.04 (C-3), 129.51 (Ar-C), 130.22 (Ar-C), 130.67 (C-2), 131.35 (C-11), 131.82 (Ar-C),
132.42 (Ar-C), 147.87 (Ar-C), 150.52 (Ar-C), 156.11 (C-9), 157.14 (C-5). IR (KBr 1%) νmax
=
3431, 3173, 2927, 2853, 1640, 1588, 1547, 1494, 1454, 1432, 1384, 1322, 1272, 1254, 1234,
1181, 1160, 1147, 1118, 1000, 948, 854, 830 cm-1. HRMS (FAB) m/z: calcd for C27H34N3O
[M+]: 416.26964; found: 416.26896.
2.3.3. N-(5-(Propylamino)-9H-benzo[a]phenoxazin-9-ylidene)dodecan-1-aminium chloride 9
The product of the reaction of 5-(dodecylamino)-2-nitrosophenol hydrochloride 4 (0.515 g, 1.5
mmol) with N-propylnaphthalen-1-amine 5 (0.139 g; 0.75 mmol) (reflux time 10 h) was
7

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chromatographed with chloroform and chloroform/methanol (mixtures of increasing polarity),
as the eluent, to give compound 9 (0.229 g, 30%). Mp = 151-153 °C. Rf = 0.46
1
(chloroform/methanol, 9:1). H NMR (CDCl3, 400 MHz):
δ
= 0.88 (t, J = 7.2 Hz, 3 H,
NH(CH2)11CH3), 1.10 (br s,
3
H, NHCH2CH2CH3), 1.26-1.43 (m, 18 H,
NHCH2CH2(CH2)9CH3), 1.76 (br s, 2 H, NHCH2CH2(CH2)9CH3), 1.95 (br s, 2 H,
NHCH2CH2CH3), 3.06 (br s, 2 H, NHCH2CH2(CH2)9CH3), 3.54 (br s, 2 H, NHCH2CH2CH3),
6.21 (s, 2 H, 8-H, 6-H), 7.13 (br s, 1 H, 10-H), 7.36 (br s, 1 H, 11-H), 7.77 (br s, 2 H, 3-H, 2-
H), 8.23 (br s, 1 H, NH), 8.67 (br s, 1 H, 4-H), 8.86 (br s, 1 H, 1-H), 10.37 (br s, 1 H, NH). 13C
NMR (CDCl3, 100.6 MHz): δ = 11.67 (NHCH2CH2CH3), 14.09 (NH(CH2)11CH3), 22.07
(2×CH2), 22.65 (NHCH2CH2CH3), 27.21 (2×CH2), 28.53 (NHCH2CH2(CH2)9CH3), 29.32
(CH2), 29.57 (CH2), 29.61 (2×CH2), 29.63 (CH2), 31.88 (CH2), 44.00 (NHCH2CH2CH3), 52.01
(NHCH2CH2(CH2)9CH3), 92.60 (C-6), 94.32 (C-8), 123.39 (C-10), 123.88 (C-4), 125.42 (Ar-
C), 129.62 (C-1), 130.22 (2×Ar-C), 130.72 (C-3), 131.47 (C-2), 131.75 (C-11), 132.52 (Ar-C),
147.81 (Ar-C), 150.50 (Ar-C), 155.93 (C-9), 157.17 (C-5). IR (KBr 1%): νmax 3159, 2923,
2850, 1643, 1585, 1548, 1519, 1494, 1450, 1434, 1416, 1384, 1317, 1303, 1286, 1271, 1254,
1235, 1186, 1171, 1160, 1145, 1119, 1097, 1056, 1014, 999, 975, 951, 855, 828 cm-1. HRMS:
m/z (FAB): calcd. for C31H42N3O [M+]: 472.33224; found: 472.33105.
2.4. Preparation of DNA mother solution
Natural double-stranded salmon sperm DNA was obtained from Invitrogen as a 10 mg/mL
aqueous solution with an average size of 2000 bp. Mother solutions of salmon sperm DNA
were made in 10 mM Tris-HCl buffer (pH = 7.4), with 1 mM EDTA. The purity of DNA was
checked by monitoring the absorption spectrum and the ratio of the absorbance at 260 and 280
nm, A260/A280 = 1.95 (good-quality DNA has an A260/A280 ratio higher than 1.8) [24]. The DNA
concentration in number of bases (or phosphate groups, [P]) were determined from the molar
extinction coefficient
ε M
= 6600 M-1cm-1 at 260 nm [25]. Appropriate amounts of 10-3
ethanolic solutions of the compounds were added to DNA solutions at desired concentrations.
The solutions were left at least 24 h to stabilize.
2.5. Electrophoresis
The electrophoretic apparatus was a BIO RAD Power Pac 300 run at an electric potential
difference of 100V. The distance between the electrodes in the electrophoretic chamber was 20
cm such that the applied electrical field was 5V/cm.
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2.5.1. Typical procedure for electrophoresis experiments
Solutions of benzo[a]phenoxazinium chlorides 6-9 (D) in DNA with molar ratio P/D = 5 were
prepared using 12 µL of salmon sperm DNA in Tris-buffer solution, [P] = 0.0025 M, 6 µL of 1
mM solution of compounds 6-9 in ethanol, 10 µL of glycerine/water 1:1 as sample loading
buffer and 32 µL of TAE buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA). A volume of
40 µL of the resulting solution was loaded in wells obtained after gelification of a 0.3%
solution of agarose gel in TAE buffer. The amount of DNA per well was 6 µg. In regular time
intervals the electrophoresis run was interrupted and images captured with a digital camera.
3. Results and discussion
3.1. Synthetic methods
Condensation reaction of 5-(diethylamino-2-nitroso)phenol hydrochloride 1 [18], 5-
(dioctylamino)-2-nitrosophenol hydrochloride 2, 2-nitroso-5-(octylamino)phenol hydrochloride
3 and 5-(dodecylamino)-2-nitrosophenol hydrochloride 4 with N-propylnaphthalen-1-amine 5
[22], in acid medium under reflux in methanol followed by purification through column
chromatography on silica gel, gave benzo[a]phenoxazinium chlorides 6 (49%), 7 (35%), 8
(78%) and 9 (30%), respectively (Scheme 1). 2-Nitrosophenol hydrochlorides 1-4 were
obtained by nitrosation of 3-(diethylamino)phenol, 3-(dioctylamino)phenol, 3-
(octylamino)phenol and 3-(dodecylamino)phenol, with sodium nitrite and hydrochloric acid, in
a mixture of ethanol-water as the solvent [23]. The latter precursors resulted from the
alkylation of 3-aminophenol with the appropriate bromoalkane, namely bromoethane, 1-
bromooctane and 1-bromododecane.
<Scheme 1>
3.2. Physical studies
Electronic absorption and emission spectra of 2×10-6 M solutions in degassed absolute
ethanol and in physiological simulated conditions (pH 7.4, Tris-HCl buffer solutions) were
measured for the synthesised benzo[a]phenoxazinium chlorides 6-9 (Table 1). The
fluorescence quantum yields (ΦF) were determined using Oxazine 1 as a standard (ΦF = 0.11 in
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ethanol) [3] at 570 nm excitation. As in previous studies with heterocycles of the same family
[17], the photophysical behaviour is determined by the coexistence of an acidic cationic form
of benzo[a]phenoxazinium chlorides (BzH+) and
a
neutral basic form of
benzo[a]phenoxazinium chlorides (Bz). The acidic form shows maximum absorption
wavelength in the 627-647 nm range and an emission in the 632-683 nm spectral region
whereas the neutral form has maximum absorption near 500 nm and a broad emission centred
at ~600 nm with a ~10 times lower fluorescence quantum yield. In water it was previously
reported that Bz neutral form is absent and non-emissive H-aggregates appeared with an
approximately 50 nm blue shifted absorption relative to the observed for the acidic form. This
aggregation resulted in a decrease of the fluorescence quantum yield.
In the case of the studied compounds 6-9 it is observed that only compound 6 maintains
a high fluorescence quantum yield in physiological pH indicating that the fraction of
aggregation is very small. The red shift of absorption maxima, when changing from ethanol to
physiological pH, is indicative of a π-π* electronic transition.
<Table 1>
In compounds 7-9, significant aggregation occurs with broad bands appearing near 580
nm and a huge decrease in fluorescence quantum yields. Comparing compounds 8 and 9, it is
seen that the increase of side-chain length on the 9-amino moiety results in an increased
fraction of aggregation and blue shift of the emission from 650 to 632 nm.
Compound 7 with a dioctylated amine shows the highest level of aggregation and evidence for
the presence of the neutral Bz form probably stabilized by an ethanol enriched solvation shell
(the preparation of all aqueous buffered solutions proceeded by addition of a 1 mM mother
solution of the compounds in ethanol) .
As a continuation of our previous studies [21,22] concerning the use of
benzo[a]phenoxazinium derivatives as DNA non-covalent markers, absorption and emission
spectra were obtained as a function of DNA phosphate group to compound molar ration (P/D).
In Figures 1 to 4, area normalized absorption spectra, as well as normalised fluorescence
spectra, are shown for compounds 6-9.
The behaviour of compound 6, which is similar to Nile Blue, was already published in a
previous study [21] and, for completeness, its results are replotted in this work (Figure 1).
Briefly, for low P/D values a decrease in absorption is observed with an appearance of a blue
shifted shoulder and a corresponding decrease in fluorescence quantum yield maintaining the
spectral shape and position. These results were interpreted as electrostatic binding that
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promotes formation of H-aggregates. For higher P/D values (>50), distinct photophysical
behaviour occurs: a red shift of both absorption and emission spectra with a slight increase in
the fluorescence quantum yield. This behaviour was interpreted [21] and proved [22] to
indicate dye intercalation into the DNA double strand.
< Figure 1>
For dyes 7-9 very different behaviour (Figures 2-4) were observed. The dialkylated
derivative 7 mainly showed variation in the fraction of basic neutral form and a decrease in
aggregation. The corresponding fluorescence intensity decreases as the quantum yield of the
neutral form is much lower than that of the cationic form. This could result from groove
binding [22,23]. For P/D>50, a red shift is observed in fluorescence spectra with an increase in
quantum yield (Figure 3). This behaviour could indicate intercalation but the noise level of the
experimental spectra does not allow a definite conclusion.
< Figure 2>
In absorption, dye 8 shows a similar behaviour to dye 6 (Figure 3). The only difference
is that aggregates exist in the absence of DNA and consequently the absorption is lower, as
well as the fluorescence quantum yield. However, in the emission behaviour, while there is a
decrease in fluorescence quantum yield, a red shift is also observed (Figure 3B). As a result
either the formed aggregates are distorted originating a red J-aggregate type emission, or
another type of DNA binding is operative. In this last case, it should be groove binding as,
similarly to compound 6, for higher P/D values (>50) the quantum yield rises and both
absorption and emission spectra shifts to the red. These last observations correspond to
intercalative binding.
In a previous study [21], a similar compound possessing the octyl group on the 5-amino
unit instead of in the 9-position showed a similar behaviour, although the red shift in emission
spectra was only observed when the dye intercalates (high P/D values). From this fact, it can be
concluded that these type of compounds can either intercalate DNA through the fused benzene
ring into the phenoxazine moiety or enter the DNA double strand using the molecular side that
is less constrained.
< Figure 3>
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Compound 9 is similar to 8 but possesses a dodecyl group on the 9-amino group, yet a
different photophysical behaviour is observed as a result of DNA interaction (Figure 4). It was
previously verified [22] that apolar/non-functionalised long side chains hinder the intercalation
of the benzo[a]phenoxazinium unit. For compound 9, no red shift in absorption is observed.
Instead, a blue shift occurs for P/D=1 after which the spectral shape remains constant with an
initial fast increase in absorption intensity followed by a slow one after P/D≥10. In emission
the spectral position and shape remains approximately constant with a decrease in quantum
yield up to P/D=5, above which there is a red shift of the spectrum with no appreciable change
in quantum yield. Up to P/D=5, the behaviour is compatible with H-aggregation, but in a local
environment different from what is experienced in the case of compound 8. Above P/D=10, the
gradual red shift in emission spectra is similar to that observed for compound 8, but the
interpretation of the emission spectra is not straightforward because, as the quantum yield is
very low, the noise level is high and increases with wavelength, as the photomultiplier
gradually loses efficiency as its detection limit of 800 nm approaches.
< Figure 4>
In order to access the potential of dyes 6-9 as DNA markers in agarose gel
electrophoresis assays and to further assess the strength of the binding of the compounds with
DNA, Figures 5 and 6 show results obtained from electrophoresis runs performed at a P/D ratio
of 5 for which the interaction with DNA is dominated by electrostatic and groove binding. The
standard procedure used in this type of assay is ethidium bromide dispersed in the agarose gel
and interacting with DNA via intercalation. This compound is very carcinogenic and suitable
alternatives are of interest. In the present experimental setup, only DNA is stained resulting in
much lesser use of the synthetic compound. The curves shown in Figures 5 and 6 were
obtained by transforming the image, taken with a digital camera after fixed periods of time,
into a gray scale and averaging the pixel intensity (0-dark; 255-white) in each lane as a
function of the distance from the wells where the DNA solutions were initially placed. The
baseline was subtracted from each curve to allow a better visualization.
< Figure 5>
The migration of DNA to the positive pole was seen as either blue (compounds 6, 8, 9)
or orange (compound 7) travelling bands. This confirms the binding of the compounds to
DNA, as migration of free compounds would have occurred in the opposite direction towards
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the negative pole. The bands faded after 30 min, but during this period they were clearly seen
by the unaided eye.
DNA lanes with compounds 6, 8 and 9 showed similar migrating velocities whereas
DNA marked with compound 7 migrated more slowly. As the bands are migrating a tail is left
on the gel. This tailing implies that some fraction of the dye leaves DNA and either starts
migrating to the opposite side or spreads within the gel. Both of these processes account for the
fading of the DNA bands. The released molecules are expected to be those that were
electrostatically binded as the applied electric field weakens this interaction. Compound 6
shows a lower amount of this tracing tail, as can be seen in the curves in Figure 5, by a lower
intensity before the peak corresponding to the DNA band. This tail can be a hindrance when
trying to separate mixtures of DNA chains with different sizes according to different migration
rates. Still, we expect that these compounds, especially 6, to be useful in such assays, as the
DNA band remains more intense than the background and also because the released dye from
the faster moving DNA chains could rebind larger DNA sequences that migrate more slowly.
Nile blue, which is similar to compound 6, has already been shown to be useful in DNA
separations [26] although the experimental setup involved marking all the gel, use of higher
electrical fields (17V/cm compared with 5V/cm used in this work) and an induction time of 15
min, that probably is necessary in order to concentrate the dye, travelling in opposite direction
to DNA, around the moving DNA spots. As a preliminary experiment to show whether there is
sensitivity to the size of the DNA strand, an electrophoresis assay was performed with the same
type of DNA but downsized using ultrasonication (QSonica Misonix S-4000 with 80W
irradiation during 1 h). Figure 6 shows the obtained results for compounds 6, 8 and 9. It can be
seen that the migration velocities are higher as expected for a smaller sized DNA chain.
Compound 6 migrates slightly faster than the other compounds and almost without a tail. For
compounds 8 and 9 the tail is very prominent and inhomogeneous.
The results for compounds 7, 8 and 9 indicates that the free compound might locally
change the agarose-gel structure due to its hydrophobic moieties leading to changes in
migration velocity of DNA and in gel homogeneity. These electrophoresis experiments also
showed that compound 7 which, from spectroscopy data, seemed to have a weak interaction
with DNA, also binds to DNA although not so strongly as the other compounds.
< Figure 6>
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4. Conclusion
Benzo[a]phenoxazinium chlorides either mono- (octyl and dodecyl groups) or
disubstituted (ethyl and octyl groups) at the 9-position of the tetracyclic ring, and having the
propyl group at the 5-amino position were synthesized. Through photophysical behavior at
varying P/D molar ratio and agarose gel electrophoresis experiments it was concluded that all
compounds interact with DNA. While compounds 6 and 8 clearly showed intercalative
behavior, the more hydrophobic derivatives 7 and 9 mainly showed either aggregation on the
surface of the DNA or groove binding. Combining the results of this contribution with previous
studies [22] it is possible to conclude that the benzo[a]phenoxazinium intercalates DNA or
from the phenoxazinium side that is less constrained or in a slating orientation through the
fused benzene ring. The compounds, especially compounds 6 and 8, also showed promising
capabilities as DNA coloured markers in electrophoresis assays without the need for staining
the gel.
Acknowledgments
Thanks are due to the Fundação para a Ciência e Tecnologia (FCT, Portugal) for
financial support to the NMR portuguese network (PTNMR, Bruker Avance III 400-Univ.
Minho), FCT and FEDER (European Fund for Regional Development)-COMPETE-QREN-EU
for financial support to Research Centres CQ/UM [PEst-C/QUI/UI0686/2011 (FCOMP-01-
0124-FEDER-022716)] and CFUM [PEst-C/FIS/UI0607/2011 (F-COMP-01-0124-FEDER-
022711)]. Post-doctoral grants to B. R. Raju (SFRH/BPD/62881/2009) and S. Naik
(SFRH/BPD/37840/2007) are also acknowledged to FCT, POPH-QREN, FSE.
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CAPTIONS
Table 1. UV-Vis-NIR absorption and fluorescence data for 2×10-6 M solutions of compounds
6-9 in ethanol and buffered (pH = 7.4) aqueous solutions.
Scheme 1. Synthesis of benzo[a]phenoxazinium chlorides 6-9.
Figure 1. Area normalised absorption spectra (left panel) and normalised fluorescence
intensity at 570 nm excitation (right panel) of compound 6 in buffered (pH = 7.4) aqueous
solutions of DNA. The curves are identified by the corresponding P/D value. The insets show
either the area of the absorption spectra (left panel) or the fluorescence quantum yield as a
function of P/D (right panel).
Figure 2. Area normalised absorption spectra (left panel) and normalised fluorescence
intensity at 570 nm excitation (right panel) of compound 7 in buffered (pH = 7.4) aqueous
solutions of DNA. The curves are identified by the corresponding P/D value. The insets show
either the area of the absorption spectra (left panel) or the fluorescence quantum yield as a
function of P/D (right panel).
Figure 3. Area normalised absorption spectra (left panel) and normalised fluorescence
intensity at 570 nm excitation (right panel) of compound 8 in buffered (pH = 7.4) aqueous
solutions of DNA. The curves are identified by the corresponding P/D value. The insets show
either the area of the absorption spectra (left panel) or the fluorescence quantum yield as a
function of P/D (right panel).
Figure 4. Area normalised absorption spectra (left panel) and normalised fluorescence
intensity at 570 nm excitation (right panel) of compound 9 in buffered (pH = 7.4) aqueous
solutions of DNA. The curves are identified by the corresponding P/D value. The insets show
either the area of the absorption spectra (left panel) or the fluorescence quantum yield as a
function of P/D (right panel).
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Figure 5. Agarose gel electrophoresis assay of salmon sperm DNA marked with compounds 6-
9. In each picture, two white lines mark the position of the wells, the order of the compounds in
the lanes are, from top to bottom, in same order as in the legend of the graph. Top figure after
10 min run. Bottom figure after 20 min run. The graph plots the pixel intensity in gray scale (0-
black; 255-white) for each lane as a function of distance from the well.
Figure 6. Agarose gel electrophoresis assay of ultrasonicated Salmon sperm DNA marked
with compounds 6, 8 and 9. In each picture, two white lines mark the position of the wells, the
order of the compounds in the lanes are, from top to bottom, in same order as in the legend of
the graph. Top figure after 5 min run. Bottom figure after 20 min run. The graph plots the pixel
intensity in gray scale (0-black; 255-white) for each lane as a function of distance from the
well.
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TABLE
Table 1
Ethanol
Tris-HCl buffer solution (pH = 7.4)
Compound
λabs (nm)
λem (nm)
λabs (nm) (a)
(104 M-1cm-1)
642
λem (nm)
ΦF
ΦF
ε
(104 M-1cm-1)
507; 637
ε
669
0.26
676
0.16
645
0.38
645
0.35
683
0.25
6
7
8
9
1.69; 2.82
514; 641
3.1
521; 610 sh; 644
0.46; 0.34; 0.40
530 sh; 588; 647 sh
0.7; 1.0; 0.8
646
1.80; 2.91
506/627
0.0063
650
1.60; 3.47
510; 628
0.052
632
583; 644 sh
1.65; 4.27
1.0; 0.68
0.018
(a) sh: shoulder
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Scheme 1
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Figure 1
21

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Figure 2
22

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Figure 3
23

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Figure 4
24

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Figure 5
25

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Figure 6
26